Integration of Three-Phase Microelectroextraction Sample Preparation Into Capillary Electrophoresis
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Journal of Chromatography A 1610 (2020) 460570 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Integration of three-phase microelectroextraction sample preparation into capillary electrophoresis ∗ Amar Oedit a, Bastiaan Duivelshof a, Peter W. Lindenburg a,b, , Thomas Hankemeier a a Division of Systems Biomedicine and Pharmacology, Leiden Academic Centre for Drug Research, Einsteinweg 55, 2300 RA Leiden, the Netherlands b University of Applied Sciences Leiden, Faculty Science & Technology, Research Group Metabolomics, Mailbox 382, 2300 AJ, Leiden, the Netherlands a r t i c l e i n f o a b s t r a c t Article history: A major strength of capillary electrophoresis (CE) is its ability to inject small sample volumes. However, Received 11 July 2019 there is a great mismatch between injection volume (typically < 100 nL) and sample volumes (typically Revised 25 September 2019 20–1500 μL). Electromigration-based sample preparation methods are based on similar principles as CE. Accepted 25 September 2019 The combination of these methods with capillary electrophoresis could tackle obstacles in the analysis of Available online 26 September 2019 dilute samples. Keywords: This study demonstrates coupling of three-phase microelectroextraction (3PEE) to CE for sample Electromembrane extraction preparation and preconcentration of large volume samples while requiring minimal adaptation of CE Electroextraction equipment. In this set-up, electroextraction takes place from an aqueous phase, through an organic filter Sample preparation phase, into an aqueous droplet that is hanging at the capillary inlet. The first visual proof-of-concept for Capillary electrophoresis this set-up showed successful extraction using the cationic dye crystal violet (CV). The potential of 3PEE Sample enrichment for bioanalysis was demonstrated by successful extraction of the biogenic amines serotonin (5-HT), tyro- Electrophoresis sine (Tyr) and tryptophan (Trp). Under optimized conditions limits of detection (LOD) were 15 nM and 33 nM for 5-HT and Tyr respectively (with Trp as an internal standard). These LODs are comparable to other similar preconcentration methods that have been reported in conjunction with CE. Good linearity ( R 2 > 0.9967) was observed for both model analytes. RSDs for peak areas in technical replicates, interday and intraday variability were all satisfactory, i.e., below 14%. 5-HT, Tyr and Trp spiked to human urine were successfully extracted and separated. These results underline the great potential of 3PEE as an inte- grated enrichment technique from biological samples and subsequent sensitive metabolomics analysis. ©2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ) 1. Introduction acceptor phase and thereby leads to stacking and preconcentra- tion of analytes. Electromigration-based techniques offer several Sample preparation is a crucial aspect of bioanalysis. The main advantages over partitioning-based techniques, such as being able objectives of sample preparation are to purify and enrich analytes to handle small sample volumes, enhanced extraction speeds (due prior to separation and detection. Commonly used sample prepa- to the electric field being the driving force rather than partitioning ration techniques are protein precipitation and partitioning based between phases) and ease of automation [4–6] . techniques, e.g., solid phase extraction (SPE) and liquid–liquid The combination of electromigration-based sample pretreat- extraction [1,2] . In the past years, electromigration-based extrac- ment with CE offers two main benefits. First, both approaches are tion methods have gained increased attention [3–5] . The main based on electromigration, so compounds that can be extracted are principle behind electromigration-based techniques is the use of also suited for CE separation. Second, electromigration-based tech- an electric field to extract ions from a donor phase (optionally niques can help overcome one of the drawbacks of CE: there is a through intermediate phases) to an acceptor phase. The migration great mismatch between the injected volume (typically < 100 nL) speed depends on the electrophoretic mobility of the analyte and and the sample volume (typically 20–1500 μL). In order to over- the electric field. The electric field strength is typically low in the come this mismatch miniaturized inserts have been developed [7] . However, when samples are too dilute and compounds fall below detection limits this does not provide a solution. Electromigration- ∗ Corresponding author. based sample preparation can help overcome the mismatch that E-mail addresses: [email protected] , [email protected] (P.W. Lindenburg). is often present between injection volumes and sample volumes https://doi.org/10.1016/j.chroma.2019.460570 0021-9673/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ) 2 A. Oedit, B. Duivelshof and P.W. Lindenburg et al. / Journal of Chromatography A 1610 (2020) 460570 in CE analysis and can offer significant advantages in loadability Previously, we have reported 3PEE as a powerful preconcentra- compared to in-line stacking methods. On-line SPE-CE, where SPE tion technique. Herein, we propose integration of this system into is coupled to the capillary, could also serve as a solution for en- a CE set-up for on-line sample clean-up and preconcentration. Ra- hancing the loadability, but has thus far only been demonstrated terink et al. demonstrated the extraction of acylcarnitines from a for more apolar compounds on apolar cartridges [8] . Moreover, to 50 μL donor phase, through a filter phase into a 2 μL acceptor the best of our knowledge, no commercial solutions for SPE-CE are phase that was formed by a conductive pipet tip prior to direct available and setting them up is complex. analysis with high-resolution mass spectrometry. In 3PEE, altering In electromembrane extraction (EME) a supported liquid mem- the organic filter phase composition influences the selectivity and brane (SLM) is used to separate the donor from the acceptor phase. enables selective extraction of desired analytes [21] . The analytes are extracted through the SLM into the acceptor In this article, we demonstrate a proof-of-principle of a novel phase using an electrical potential. Several set-ups in which EME on-line analytical system in which electromigration-based sample was coupled off-line to CE-UV and applied to bioanalysis have been preparation technique, i.e., 3PEE, is directly hyphenated to CE-UV. reported [6,9–15] . Our method offers several advantages: automation, sample precon- Off-line, at-line, as well as in-line coupling of EME to CE-UV centration and the ability to extract analytes from salt-rich ma- has been reported on several occasions. An off-line process con- trices without sample preparation. In order to enable 3PEE the sists of two steps (e.g. sample preparation followed by separation). electrode configuration of a commercially available CE apparatus An at-line process combines the two steps via an automated was modified. This modification entails placing an insulating sleeve handler (e.g. a robot). An in-line process takes place within the over the electrode whilst leaving the bottom part of the electrode separation system (e.g. SPE-CE with sorbent inside the capillary). exposed. 3PEE-CE-UV permits automated on-line selective analyte An on-line process takes place right before the sample stream extraction and enrichment, directly from a dilute sample. In this enters the separation system, but does take place within the set-up the analytes are extracted from a sample vial containing a analytical instrument (e.g. SPE-CE with sorbent outside the cap- two-phase liquid-liquid system, i.e., the aqueous sample and an illary) [16] . An example of on-line coupling of EME to CE-UV, is organic filter phase. Extraction takes place into a droplet of ac- nano-EME, for preconcentration and analysis of drugs. Here, basic ceptor phase hanging at the capillary inlet in the organic filter drugs were extracted from a sample volume of 200 μL, through a phase. membrane over a cracked capillary, into an acceptor of ∼8 nL. A In a first series of experiments the process was visualized us- single SLM could last for more than 200 extractions. Enrichment ing the cationic dye CV to assess the stability of the droplet of ac- factors (EFs) ranging between 25 and 196 were reported for basic ceptor phase during 3PEE. Then, the proposed method was eval- drug compounds, corresponding to recoveries of 0.1% and 0.79%. uated using selected biogenic amine model compounds to evalu- Under different conditions, higher EFs were obtained, up to 500 ate its potential for bioanalysis of polar metabolites. In order to for loperamide [17] . Moreover, EME-CE for preconcentration and enhance the CE separation, pH-mediated stacking was included analysis of basic drugs was reported. Here, an SLM was formed in the system. Consecutively, important extraction parameters (EE between two conical polypropylene units and extraction took voltage and EE time) and selectivity of the developed 3PEE-CE- place from the device, which was used as a vial insert. Extrac- UV method were investigated to optimize extraction. Then, the tion took place from a 40 μL donor compartment over the SLM analytical performance of 3PEE-CE-UV was compared to conven- into an acceptor compartment of 40 μL and therefore it only tional CE-UV. Finally, the performance of 3PEE-CE-UV as a sam- served for sample cleanup and not for sample preconcentration. ple preparation procedure for polar compounds in salt-rich bi- Recoveries ranging from 37% to 84% were obtained [18] . However, ological samples was investigated by analysis of spiked human the device requires reassembly for each new experiment, which urine samples in order to show its applicability for metabolomics hampers automated analysis.